Fluorescence imaging techniques play a pivotal role in our understanding of the nervous system. The emergence of various super-resolution microscopy methods and specialized fluorescent probes enables direct insight into neuronal structure and protein arrangements in cellular subcompartments with so far unmatched resolution. Super-resolving visualization techniques in neurons unveil a novel understanding of cytoskeletal composition, distribution, motility, and signaling of membrane proteins, subsynaptic structure and function, and neuron-glia interaction. Well-defined molecular targets in autoimmune and neurodegenerative disease models provide excellent starting points for in-depth investigation of disease pathophysiology using novel and innovative imaging methodology. Application of super-resolution microscopy in human brain samples and for testing clinical biomarkers is still in its infancy but opens new opportunities for translational research in neurology and neuroscience. In this review, we describe how super-resolving microscopy has improved our understanding of neuronal and brain function and dysfunction in the last two decades.